QC Report


general
Report generated at2025-02-25 03:26:50
TitleAdrenalGland
DescriptionATAC-seq data from Human ENCODE adrenal gland data processed in 2025
Pipeline versionv2.2.0
Pipeline typeatac
Genomehg38
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads537908128145594466
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads527417446139999369
Mapped Reads (QC-failed)00
% Mapped Reads98.096.2
Paired Reads537908128145594466
Paired Reads (QC-failed)00
Read126895406472797233
Read1 (QC-failed)00
Read226895406472797233
Read2 (QC-failed)00
Properly Paired Reads516592006137227690
Properly Paired Reads (QC-failed)00
% Properly Paired Reads96.094.3
With itself521534888138536038
With itself (QC-failed)00
Singletons58825581463331
Singletons (QC-failed)00
% Singleton1.09999999999999991.0
Diff. Chroms518121124955
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads23208845261152820
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads485752809574816
Paired Optical Duplicate Reads1070803589818
% Duplicate Reads20.929615.6572

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads483685414130786411
Rm = Number of Mitochondrial Reads474333609841071
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.08930838509579780.06997971420692863

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads364041376101578580
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads364041376101578580
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads364041376101578580
Paired Reads (QC-failed)00
Read118202068850789290
Read1 (QC-failed)00
Read218202068850789290
Read2 (QC-failed)00
Properly Paired Reads364041376101578580
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself364041376101578580
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.44068825602021920.4105701927246308
Fraction of reads in NFR (QC pass)TrueTrue
Fraction of reads in NFR (QC reason)OKOK
NFR / mono-nuc reads1.9855760296730171.7470083255726019
NFR / mono-nuc reads (QC pass)FalseFalse
NFR / mono-nuc reads (QC reason)out of range [2.5, inf]out of range [2.5, inf]
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakTrueTrue

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.60010496169534310.5810448620171693
Fraction of Reads in blacklist regions0.00056247727181428960.0006207411050636857
Fraction of Reads in promoter regions0.263872066014825770.2909541854198001
Fraction of Reads in enhancer regions0.420233767603383660.37593000414063676

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments21516815357640598
Distinct Fragments18551607751090993
Positions with Two Read179507704915230
NRF = Distinct/Total0.8621910.886372
PBC1 = OneRead/Distinct0.8800020.889522
PBC2 = OneRead/TwoRead9.0945689.246073

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt239704150470
N1280753203082
N2234589154780
Np282143206765
N optimal282143206765
N conservative239704150470
Optimal Setpooled-pr1_vs_pooled-pr2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio1.177047525281181.374127733102944
Self Consistency Ratio1.1967867206049731.312068742731619
Reproducibility Testpasspass

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks299709289749

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile303.0262.0521.0349.0
50 percentile (median)582.0506.0855.0656.0
75 percentile1028.0966.01281.01120.0
Max size4503.03555.04126.04126.0
Mean727.200017350163671.8459459739291941.6516044785143791.3609162729538

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment19.18935193397206620.38705397658804

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.110442216837955660.11479228523280666
Synthetic AUC0.49802443809636650.4963040114308088
X-intercept0.096769033909149070.14057301663467692
Synthetic X-intercept0.00.0
Elbow Point0.86454134676903390.846521113243762
Synthetic Elbow Point0.50054746264118160.5059644289629819
Synthetic JS Distance0.61326542109245860.5879271674713874

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.57501126465360910.51110330544096990.56385100577138790.49570588602439610.56906527020708760.497119550204383640.56490381395938280.55384550571109970.5591735849053686

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.52982565678520880.56703923127683150.489852309414051670.5578980167250391

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.453580082379458850.52334593142511370.44175206032610420.5173627094282016

For macs2 raw peaks:


For overlap/IDR peaks: